WO2019052521A1

WO2019052521A1 - Integrated tubular reaction device

Info

Publication number: WO2019052521A1
Application number: PCT/CN2018/105633
Authority: WO
Inventors: 苏星; 吴开原
Original assignee: 星源智（珠海）生物科技有限公司
Priority date: 2017-09-18
Filing date: 2018-09-14
Publication date: 2019-03-21
Also published as: CN107523487A; US11565233B2; US20200215512A1; WO2019052521A9; CN107523487B

Abstract

An integrated tubular reaction device comprises a reaction vessel (1), a cover body (2), and a seal member (3). The reaction vessel (1) comprises at least two tubular chambers (11), a channel (12) in communication with the at least two tubular chambers (11), and an opening (13). The cover body (2) can match the opening (13) and comprises a through hole (21). The seal member (3) comprises a seal plug (31) capable of matching the through hole (21). The integrated tubular reaction device resolves the problem of reaction product pollution in processes of multiple or multi-step biological enzyme reactions, so that multiple or multi-step biological enzyme reactions can be implemented in a same device.

Description

Integrated tubular reaction device

Technical field

The present invention relates to the field of biochemical reaction devices, and in particular to an integrated tubular reaction device capable of performing a plurality of simultaneous or continuous multi-step reactions and capable of blocking the reaction device and preventing product contamination.

Background technique

With the development of biotechnology, modern molecular biology technology or genetic engineering technology is being widely used in various biotechnology industries, especially in medical diagnosis. The application of such techniques often involves multiple or sequential multi-step enzymatic reactions. For example, when it is necessary to detect an RNA virus, it is first necessary to purify the viral RNA, and then reverse-transcribe the RNA into cDNA, and finally perform a cDNA amplification reaction. A typical DNA (or cDNA) amplification reaction is the polymerase chain reaction, PCR. In a similar multi-step enzymatic reaction, the prior art requires separate steps for each step, specifically, each reaction is carried out in a separate separate tube, operated by hand or robot.

However, when a separate separation tube is used for a multi-step reaction, during the process in which the reaction product is transferred from one tube to another, the product molecules are exposed to the outside of the tube, which tends to cause contamination of the working environment by the product molecules, leading to the future. A false positive reaction occurred in the sample test. Moreover, the use of separate separate test tubes for multi-step reactions takes up a lot of space and takes time and material, thereby increasing the cost of use.

In order to more conveniently carry out multiple or continuous multi-step enzymatic reactions, the prior art also provides a new technical solution, using microfluidic technology to integrate different steps to achieve the purpose of automation. Products created with microfluidic technology are often referred to as integrated biochips. The use of integrated biochips can avoid product molecular contamination, but its structure is complex and the manufacturing cost is high. In addition, the integrated biochip has a small structure that does not match the volume of a typical biological sample. Therefore, the practical value of integrated biochips is low.

technical problem

In order to solve the above problems, it is an object of the present invention to provide an integrated tubular reaction apparatus which can be used for a plurality of or continuous multi-step reactions, which can avoid product contamination and has practical value.

Technical solution

In order to achieve the above main object, the present invention provides an integrated tubular reaction apparatus comprising: a reaction vessel comprising at least two tubular chambers, a passage connecting at least two tubular chambers, and an opening; a cover body and a cover body The closure can be closedly engaged with the opening, the cover body includes a through hole, and the sealing member includes a sealing plug that can cooperate with the through hole.

The integrated tubular reaction device of the invention is a closed whole body mainly formed by a reaction container, a cover body and a sealing member, which can effectively avoid contamination of the reaction product and prevent false positive reaction. The reaction vessel and the lid can be closed by any existing sealing method.

The cover body is provided with a through hole, and the through hole is closed by a sealing plug. The through hole can be used for sample loading and sampling. When sampling and loading are needed, only the through hole is opened, and the entire cover body is not needed to avoid the reaction system and the air. Large area contact; when sampling and loading are completed, sealing can be achieved by inserting a sealing plug, which is convenient to operate.

At least two of the tubular chambers may be subjected to the same or different reactions, and the tubular chambers are preferably disposed separately to avoid temperature disturbances caused by the close proximity of the tubular chambers. At the same time, at least two tubular chambers are connected by at least one channel, and the channels can be tubular or channel type, and the reaction products can be transported between the tubular chambers to achieve a multi-step reaction, for example, two or more enzymes. reaction. The transfer mode can be physical or chemical, such as molecular diffusion, convection or mechanical transfer, etc., and the multi-step biochemical enzymatic reaction can be automated.

In addition, the integrated tubular reaction device of the invention integrates a plurality of tubular chambers into one body, and has small volume, saves materials, low cost, small space occupation, easy operation and use, can shorten operation time, and generate valuable value quickly and effectively. The result is of great practical value.

A further technical solution is that the channels are arranged at the upper ends of the at least two tubular chambers; the openings are arranged at the upper ends of the channels.

The at least two tubular chambers of the present invention are connected by channels, and the connection locations may be provided at the upper, middle or lower ends of the tubular chamber. Ideally, the connection through the upper end facilitates mixing of substances between different tubular chambers where mixing is undesirable. On the basis of this, preferably, the opening is provided at the upper end of the passage, and the opening is opposed to the upper end of the tubular chamber, so that it is more convenient to load and sample the tubular chamber through the cover body engaged with the opening.

A further technical solution is that the through hole is arranged corresponding to the tubular chamber.

The number of through holes is the same as or different from the number of tubular chambers, and the positions of the through holes are functionally in one-to-one correspondence with the positions of the tubular chambers. When the cover body is engaged with the opening of the reaction container, the through holes on the cover body are in one-to-one correspondence with the tubular chamber, so that each of the tubular chambers can be separately connected or closed to the outside to prevent contamination, and the sample loading and sampling are more convenient. .

A further technical solution is that the at least one seal further comprises a sealing rod that is sealingly engageable with at least a portion of the tubular chamber. A further technical solution is that the sealing rod is fixedly or movably connected to the sealing plug.

The seal may comprise a sealing rod, the sealing rod being engageable with the tubular chamber for closing or opening the tubular chamber, each tubular chamber being independently closable or openable, enabling functional separation of the plurality of tubular chambers or connection. The sealing rod can be fixedly or movably connected to the sealing plug. For example, the sealing rod can be movably passed through the sealing plug, and when the tubular chamber needs to be opened, the sealing rod can be pulled up from the sealing plug. The sealing rod can also be used for sample collection prior to reaction.

A further technical solution is that the reaction vessel comprises an arcuate connection disposed between adjacent at least two tubular chambers and projecting into the passage.

An arcuate connection protruding into the passage between the adjacent at least two tubular chambers can avoid dead angles and facilitate the transmission and exchange of reaction products between adjacent tubular chambers.

A further technical solution is that the tubular chamber is cylindrical or conical, the inner diameter is between 0.1 mm and 10 mm, the wall thickness is between 0.05 and 5 mm; the ratio of the depth of the tubular chamber to the inner diameter is greater than Or equal to 2.

The inner diameter of the tubular chamber is between 0.1 mm and 10 mm, and the ratio of the depth to the inner diameter of the tubular chamber is greater than or equal to 2, which satisfies the needs of the general biological enzymatic reaction. The wall thickness between 0.05 and 5 mm ensures safety and stability and saves costs.

A further technical solution is that the integrated tubular reaction device is made of a transparent material.

In the closed state, the reaction results can be qualitatively or quantitatively detected by optical or electrical means. The integrated tubular reaction device is prepared by using a transparent material, and the reaction result is detected by an optical method, which is convenient and rapid.

A further technical solution is that the reaction vessel further comprises a storage chamber for placing a reagent or sample on one side of the tubular chamber.

The integrated tubular reaction device can also be provided with a storage chamber that can be spaced from the tubular chamber for placing reagents or samples for ease of loading.

A further technical solution is to place the same or different reagents in different tubular chambers; the channels are filled with one or more media.

The same or different reagents are placed in different tubular chambers to accomplish multiple reactions or multiple different reactions. The channel is filled with one or more mediums, so that the tubular chambers are functionally connected, and the medium can be used to transport the reaction products in the tubular chamber by molecular diffusion, liquid convection, and the like. The reagent can also be pre-stored in the tubular chamber, sealed and stored and transported. Only the sample to be tested can be added during use.

Beneficial effect

The integrated tubular reaction device of the invention can be used for multiple or continuous multi-step reactions, can avoid product pollution, prevent false positive reaction, and has low device cost, small space occupation and convenient operation.

DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an exploded view of the overall structure of an embodiment of the integrated tubular reaction apparatus of the present invention.

2 is a schematic view showing the structure of a reaction vessel in an embodiment of the integrated tubular reaction apparatus of the present invention.

Figure 3 is a schematic view showing the structure of the front side of the reaction vessel in the embodiment of the integrated tubular reaction apparatus of the present invention.

4 is a schematic view showing the structure of a cover body in an embodiment of the integrated tubular reaction device of the present invention.

Figure 5 is a schematic view showing the structure of a seal member in an embodiment of the integrated tubular reaction device of the present invention.

Embodiments of the invention

The integrated tubular reaction apparatus of the present invention will be further described below in conjunction with the accompanying drawings and examples.

As shown in Fig. 1, the integrated tubular reaction apparatus of the present embodiment comprises a reaction vessel 1, a lid body 2 and a sealing member 3, and a closed whole body is formed by the reaction vessel 1, the lid body 2 and the sealing member 3, thereby avoiding the reaction. The product pollutes the working environment, prevents false positive reactions, and the reagents do not pollute the outside world.

As shown in Figures 2 to 3, the reaction vessel 1 comprises at least two tubular chambers 11, which can perform different reactions in a multi-step reaction, as well as a plurality of identical reactions. The number of tubular chambers 11 can be determined according to the needs of the actual reaction. The tubular chamber 11 can be communicated by one or more passages 12 at the upper, middle or lower end of the tubular chamber 11, and the passage 12 allows the reaction products to be transported between the tubular chambers 11 under closed conditions to effect a multi-step reaction. The multi-step biochemical enzymatic reaction can be automated by means of physical or chemical means such as molecular diffusion, liquid convection or mechanical timed sampling. Specifically, in the present embodiment, the plurality of tubular chambers 11 are communicated by a passage 12 at the upper end of the tubular chamber 11, and the manner in which the upper ends communicate is advantageous to prevent unnecessarily mixing the reaction materials between the different tubular chambers 11. The channel 12 can be designed in different shapes as desired, such as tubular, canal, and the like.

In the present embodiment, the inner diameter of the tubular chamber 11 is between 0.1 mm and 10 mm, and the wall thickness is between 0.05 and 5 mm; the ratio of the depth to the inner diameter of the tubular chamber 11 is greater than or equal to two. The size of the tubular chamber 11 is within the above range, which satisfies the needs of the general multi-step biological enzymatic reaction, and is safe and stable, and saves cost.

The reaction vessel 1 further includes an opening 13 which, in the present embodiment, is located at the upper end of the passage 12, the opening 13 being opposite or substantially opposite the upper end of the tubular chamber 11. The opening 13 can be closedly engaged with the cover body 2 in any manner conventionally sealed, such as bonding. As shown in FIG. 4, the cover body 2 is provided with a through hole 21 through which the sample can be sampled and sampled. In the present embodiment, the number of through holes 21 is the same as or different from the number of the tubular chambers 11, and the two correspond in position and function. When the cover is engaged with the opening 13, the through hole 21 is located above the tubular chamber 11 to facilitate loading and sampling.

A seal 3 can be inserted into the through hole 21. Each of the through holes 21 can be engaged with a separate separate or associated seal 3 to achieve independent or uniform opening or closing of the respective through holes 21. As shown in FIG. 5, each of the seals 3 includes a sealing plug 31 engageable with the through hole 21, and the sealing plug 31 can be sealingly fitted with the through hole 21. The seal 3 can also include a sealing rod 32 that can be mated with at least a portion of the tubular chamber 11 for closing or opening the tubular chamber 11, each tubular chamber 11 being independently closable or openable.

The sealing rod 32 may be fixedly or movably coupled to the sealing plug 31. For example, the sealing rod 32 may pass through the sealing plug 31 so as to be movable up and down. When the tubular chamber 11 needs to be opened, the sealing rod 32 is pulled up from the sealing plug 31. can. The sealing rod 32 can be used for sample collection before the reaction, and the collected biological sample is sent to the reactor through the sealing rod 32 for reaction. For example, sample collection can be performed through the bottom end of the sealing rod 32. As another example, a sample rod or sample needle having a slightly smaller diameter may be disposed at the lower end of the sealing rod 32. The sample rod or sample needle preferably includes a rough surface in the radial direction for loading or sampling a small amount of sample. Alternatively, a sample rod or sample needle can be placed directly on the sealing plug 31 without the sealing rod 32 for loading and sampling. The sample rod or sample needle can be fixedly or movably coupled to the sealing plug 31. Preferably, the sample rod or sample needle can be moved up and down through the sealing plug 31 to facilitate lifting the sample rod or sample needle after the loading is completed. Preferably, the end of the sample rod or sample needle is provided with a hydrophilic surface for drawing the sample, and the hydrophilic surface may be a non-completely smooth surface.

When the sealing rod 32 is fixedly connected to the sealing plug 31, for a plurality of sealing members 3 in an integrated tubular reaction device, the partial sealing member 3 may be provided with a sealing rod 32, and the partial sealing member 3 does not have a sealing rod 32 through The communication or partitioning of the plurality of tubular chambers 11 can be achieved using the sealing member 3 with the sealing rod 32 and without the sealing rod 32. Alternatively, the communication or disconnection of the plurality of tubular chambers 11 is achieved by moving the sealing plug 31 with the sealing rod 32 up and down in the through hole 21.

Arranged between adjacent at least two tubular chambers 11 is an arcuate connecting portion 14 projecting into the passage 12, the arcuate connecting portion 14 avoiding dead angles, facilitating reaction products between adjacent tubular chambers 11 Transmission and exchange.

In the closed integrated tubular reaction apparatus of the present embodiment, the reaction results can be qualitatively or quantitatively detected by optical or electrical methods. Preferably, the integrated tubular reaction device is made of a transparent material such as plastic, glass, or the like, and thus has a transparent appearance, and the reaction result can be continuously and rapidly detected by an optical method in real time. The various components of the integrated tubular reactor can be integrally formed by machining or injection molding.

Further, in order to facilitate the placement of the reaction reagent or sample, a storage chamber 15 may be provided in the reaction vessel 1, and the storage chamber 15 may be spaced apart from the tubular chamber 11. For example, a through hole 21 may be provided at a position corresponding to the storage chamber 15 at the position corresponding to the storage chamber 15, for taking in a reagent or a sample, and sealing using the sealing member 3, thereby separating the storage chamber 15.

The apparatus of this embodiment can be applied to a plurality of simultaneous reactions, and can also be applied to a multi-step continuous reaction. When the apparatus of the present embodiment is used for a plurality of simultaneous reactions, the same or different reagents may be placed in different tubular chambers 11. When the apparatus of the present embodiment is applied to a multi-step continuous reaction, different reaction reagents can be placed in different tubular chambers 11 to complete multiple different reactions. The reaction reagent can also be pre-stored in the tubular chamber 11, sealed and stored and transported, and only the sample to be tested can be added during use. The tubular chamber 11 can also be filled with reagents and biological samples by manual or automated methods prior to use. For a biological enzyme reaction carried out under a closed condition, the reaction reagent contains an organic or inorganic substance such as an enzyme, a buffer, or a nucleic acid.

After the addition of the sample and the reaction reagent, the integrated tubular reaction device is closed, and then the temperature is controlled to carry out the reaction. The biological enzyme reaction is generally carried out between 15 ° C and 99 ° C. The currently known methods can be used to control the temperature of the biological enzyme reaction in the tubular chamber, for example, using infrared light, hot/cold wind, cold/hot solid or liquid substances, electromagnetic induction, and the like. The closed reaction unit can be inserted into a temperature control instrument for reaction. Depending on the requirements of the reaction, any tubular chamber 11 can be subjected to a constant temperature or periodic varying temperature, and there can also be a uniform temperature or gradient temperature within the tubular chamber 11. For example, similar to conventional PCR temperature control, when a temperature-controlled method with periodic average temperature is used, the temperature of the temperature control instrument is periodically changed under the control of a computer program, such as holding at a certain temperature for several seconds to several minutes. And the tubular chamber 11 is fully inserted into the heating portion of the temperature control instrument during which the temperature of the liquid within the tubular chamber 11 is substantially equal. As another example, in a constant temperature gradient temperature control method, the temperature of the temperature control instrument remains unchanged under the control of a computer program, and the tubular chamber 11 is only partially in contact with the heated portion of the temperature control instrument. When the bottom is heated, the bottom temperature will be higher than the top temperature, at which point the liquid in the tubular chamber 11 will have a temperature gradient. Since the liquid with a low upper temperature has a relatively high density or specific gravity, the upper and lower liquids will convect, and the effect is to drive the molecules in the tubular chamber to flow and to withstand different temperatures to meet different enzyme reaction conditions. The purpose of nucleic acid amplification in the tubular chamber 11 is achieved. The tubular structure of the tubular chamber 11 provides more flexibility to the instrument design.

The molecular transport between the different tubular chambers 11 can take the form of an active or passive. For example, the channel 12 may be filled with one or more media such that the tubular chamber 11 is functionally coupled to utilize the medium to effect molecular transport within the tubular chamber 11 by molecular diffusion or liquid convection. Sampling and loading can also be done physically or mechanically.

After the reaction product is transferred to another tubular chamber 11, the second step reaction can be carried out by a temperature control method similar to the above reaction.

After the reaction is completed, an optical or electrical signal related to the amount of the product is obtained by binding of the molecular probe or the affinity substance to the reaction product, thereby qualitatively or quantitatively detecting the reaction product. The optical signal includes a fluorescent signal, a light absorption signal, a red absorption signal, a Raman scattering signal, a chemiluminescence signal, and the like. After the completion of the reaction, the entire reaction apparatus can be subjected to high temperature or combustion treatment to prevent product contamination.

It is to be understood that the above is only the preferred embodiments of the present invention and is not intended to limit the present invention, and that various changes and modifications can be made in the present invention. Any modifications, equivalent substitutions, improvements, etc. made therein are intended to be included within the scope of the present invention.

Industrial applicability

It can be seen from the above that the integrated tubular reaction device of the present invention is entirely closed, and solves the problem of contamination of reaction products in the multi-step biochemical enzymatic reaction process. It can also perform multi-step biochemical enzymatic reactions in the same device, such as nested PCR reaction, RT-PCR reaction, multiplex PCR reaction, etc., and can also realize automated multi-step biochemical enzymatic reaction by means of molecular diffusion and convection. The device of the invention has low cost, small occupied space, and is easy to operate and use, and has great practical value.

Claims

An integrated tubular reaction device, comprising:

a reaction vessel comprising at least two tubular chambers, passages communicating with at least two of the tubular chambers, and openings;

a cover body, the cover body being closably engageable with the opening, the cover body comprising a through hole;

a seal comprising a sealing plug engageable with the through hole.
An integrated tubular reaction device according to claim 1 wherein:

The passage is disposed at an upper end of the at least two tubular chambers;

The opening is disposed at an upper end of the passage.
An integrated tubular reaction apparatus according to claim 1 or 2, wherein:

The through hole is disposed corresponding to the tubular chamber.
An integrated tubular reaction device according to any one of claims 1 to 3, characterized in that:

At least one of the seals further includes a sealing rod that is closably engageable with at least a portion of the tubular chamber.
An integrated tubular reaction apparatus according to claim 4, wherein:

The sealing rod is fixedly or movably connected to the sealing plug.
An integrated tubular reaction apparatus according to any one of claims 1 to 5, characterized in that:

The reaction vessel includes an arcuate connection disposed between adjacent ones of the at least two tubular chambers and projecting into the passage.
An integrated tubular reaction apparatus according to any one of claims 1 to 6, wherein:

The tubular chamber is cylindrical or conical, having an inner diameter of between 0.1 mm and 10 mm and a wall thickness of between 0.05 and 5 mm;

The ratio of the depth to the inner diameter of the tubular chamber is greater than or equal to two.
An integrated tubular reaction apparatus according to any one of claims 1 to 7, wherein:

The integrated tubular reaction device is made of a transparent material.
An integrated tubular reaction apparatus according to any one of claims 1 to 8, wherein:

The reaction vessel also includes a storage chamber on one side of the tubular chamber for placing a reagent or sample.
An integrated tubular reaction apparatus according to any one of claims 1 to 9, wherein:

Different or different reaction reagents are placed in different tubular chambers;

The channel is filled with one or more media.